A number of nitroaromatic compounds have substantial toxicity. In fact, certain of such compounds have been effectively used as herbicides (e.g., "dinoseb" (2-(1-methylpropyl)-4,6-dinitrophenol)), insecticides, and miticides. Perhaps the most notorious use of certain of these compounds is as explosives, such as 2,4,6-trinitrotoluene ("TNT"), dinitrotoluene ("DNT"), and picric acid (2,4,6-trinitrophenol). A similar group of compounds consists of the nitramines, exemplified by the "munitions" explosives hexahydro-1,3,5-triazine ("RDX") and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine ("HMX"). Contaminant nitroaromatics and nitramines are generally persistent in natural environments, where the residual toxicity of these compounds can present a substantial hazard. A number of sites worldwide are contaminated with one or more of these compounds, including both manufacturing and military sites, thereby rendering the sites unfit for other uses. Thus, there is a need for ways in which such sites can be remediated for other uses.
Heretofore, land farming has generally been employed in attempts to remove these chemicals from contaminated lands, wherein, for example, contaminated soil is augmented with fertilizer and the mixture aerated to promote microbial activity. Unfortunately, nitroaromatics and nitramines are not satisfactorily degraded by land farming or other aerobic methods. Possible reasons include lack of indigenous nitroaromatic-degrading microorganisms, partitioning of the contaminant chemicals to biologically sequestered or inhospitable parts of the environment, and accumulation of toxic partial-breakdown by-products. Problems with land farming in general include the slow rate of biodegradation, high expense, and accumulation of toxic by-products.
Other methods to remove nitroaromatics and nitramines from contaminated soils have also met with little practical success. Such methods include transportation of contaminated soil to hazardous waste dumps, and on-site incineration of the soil. Problems with such methods include high cost and poor accountability of the responsible party.
Certain nitroaromatic molecules, and possibly certain nitramines, are susceptible to "transformation" reactions in natural environments. "Transformation" (or "biotransformation" if biologically mediated) is not the same as "biodegradation." As a result of transformation, molecules of the contaminant compound are converted into other environmentally resistant compounds that retain a substantial amount of the residual toxicity of the original compound. These other compounds can have larger molecular weights than the original contaminant compounds. "Biodegradation" means the biologically mediated conversion of the contaminant compound at least to organic acids and/or other simple organic molecules that lack the residual toxicity of the original compound and that are readily metabolized by other organisms. "Mineralization" is degradation carried to the extreme, in which molecules of the contaminant compound are converted to carbon dioxide and other non-organic fundamental compounds.
For example, the anaerobic bacteria Veillonella alkalescens can reductively transform nitroaromatic compounds, converting the nitro groups to amino groups. McCormack et al., Appl. Environ. Microbiol. 31:949-958 (1976). Unfortunately, aminoaromatic derivatives of nitroaromatics tend to be transformed in the presence of atmospheric oxygen to polymeric (large molecular weight) compounds, Parris, Residue Revs. 76:1-30 (1980), that are usually incorporated in the field into long-lived soil humic matter that retains a substantial amount of the toxicity of the original compound. Channon et al., Biochem. J. 38:70-85 (1944); McCormick et al., Appl. Environ. Microbiol. 31:949-958 (1976); Simmons et al., Environ. Sci. Technol. 23:115-121 (1989).
Biodegradation of a nitroaromatic compound by a microorganism would require that the microorganism be capable of removing nitro groups and cleaving aromatic rings to alkyl groups. Biodegradation of a nitramine compound would also require an ability of the responsible microorganism to remove nitro groups. No single anaerobic strain capable of carrying out this process has been isolated to date.
In co-pending U.S. Patent application serial no. 08/096,735, incorporated herein by reference, a biological system for degrading contaminant nitroaromatics in waters and soils is disclosed. The method typically involves an aerobic stage followed by an anaerobic stage, wherein the aerobic stage is short-lived to prevent formation of recalcitrant aminoaromatic polymers from the nitroaromatics. Actual degradation, and even mineralization, of the nitroaromatic occurs during the anaerobic stage, involving multiple reactions performed by microorganisms comprising an anaerobic consortium. The consortium is preferably obtained by removing a sample of soil from the contaminated site and culturing the resident microorganisms under anaerobic conditions in the presence of the particular nitroaromatic(s) to be degraded. Over time in such conditions, the culture becomes enriched in microorganisms capable of degrading the target nitroaromatic(s). The resulting enriched culture can then be used to inoculate a quantity of soil or water to be bioremediated. Even though this method has substantial utility, it would be desirable to have various pure cultures of microorganisms each capable of completely mineralizing one or more specific nitroaromatic and/or nitramine compounds.
Thus, there remains a need for pure cultures of microorganisms capable of at least biodegrading nitroaromatics and/or nitramines, and for methods for biodegrading such compounds in which a pure culture of microorganisms can be added to contaminated soil or water for the purpose of biodegrading such compounds present as contaminants in the soil or water.
Further, there is a need for such a method that can be performed at a natural site that has soil or water contaminated with a nitroaromatic and/or nitramine compound.